Antibiotic composition

ABSTRACT

The present invention provides antimicrobial compositions comprising an antibiotic and a sensitizer that enhances the effectiveness or activity of the antibiotic, wherein the sensitizer is preferably a primary amine containing a long alkyl chain. Such compositions are particularly useful for the treatment of infections by drug resistant bacteria.

FIELD OF THE INVENTION

The present invention is in the field of antimicrobial compositions, inparticular in the field of antimicrobial compositions comprising anantibiotic and a compound that acts as an enhancer of the antimicrobialeffect of the antibiotic.

BACKGROUND OF THE INVENTION

During the last decades a dramatic increase in bacterial strains thatare resistant to one or more antibiotics has been reported. Thisincrease has led to the occurrence of bacterial infections that canhardly or not at all be treated with the existing spectrum ofantibiotics, which is a particularly serious problem in connection withe.g. hospital-acquired infections and has led to an increase inbacterial infections with a fatal outcome. The emergence of antibioticresistance appears to be a result of the incorrect use of antibiotics inhuman and veterinary medicine combined with efficient bacterial mutationmachinery.

The cell wall is a bacterial feature where particular antibioticresistance originates, as it prevents many antibiotics from reachingtheir targets inside the cell, and may contain antibiotic efflux pumpsystems. Furthermore, bacteria may produce antibiotic hydrolyzingproteins (e.g. β-lactamases) that inactivate antibiotics. It isgenerally believed that if the bacterial membrane could only be renderedmore permeable, the effect of antibiotics would be enhanced. Manyattempts have been made to find effective ways of permeabilizing thebacterial outer membrane. Several polycations have been shown topermeabilize the outer membrane of Gram-negative bacteria, presumably bybinding to the negatively charged lipopolysaccharide (LPS). Among thepolycation permeabilizers are polymyxin B and its derivatives (see forexample U.S. Pat. No. 4,510,132). Other polycationic permeabilizersinclude bactericidal/permeability-increasing protein, prolamine, andvarious polycationic and/or amphiphilic peptides including lysineoligomers, defensins, cecropins, magainins, and mellitin. Negativelycharged chelators, such as ethylenediaminetetraacetate (EDTA),nitrilotriacetate, and sodium hexametaphosphate have also proved to beeffective outer membrane permeabilizers, presumably by removing calciumand magnesium ions that cluster LPS units together, resulting inmembrane destabilization. U.S. Pat. No. 6,165,997 discloses negativelycharged phospholipids enhancing the activity of antimicrobials, but alsohaving antimicrobial activity themselves. These compounds appear to act,at least partially, as the cation chelators described above.

JP 57155954 describes the enhancement of the activity of a basic peptideantibiotic substance in feed through the presence of a lipoamineconsisting of 4 or more carbon atoms. The basic peptide antibiotic isbased on amines, e.g. colistin A or B or polymyxin A, B, D or M.Disclosed lipoamines are monoamines, in particular butylamine,pentylamine, hexylamine, heptylamine, octylamine, nonylamine,decylamine, laurylamine and stearylamine. The focus is on suppressingthe decrease of antibacterial activity caused by phospholipids,unsaturated fatty acids and calcium and magnesium present in livestockby addition of these amines to animal feed containing the antibacterialpolymyxins. JP 57155954 shows that large amounts of lipoamines arerequired to restore or even improve antibacterial activity in feed,especially with respect to the amounts of antibacterial peptides presentin the feed. Effects are only observed at molar ratios of stearylamineto colistin of at least 13:1. These relatively high concentrations oflipoamines would make them unattractive for use outside the field ofanimal feed.

Moreover, the lipoamines in JP 57155954 are carefully selected to matchlook-a-like polymyxins, resembling the hydrophobic tail which isessential for antibacterial activity itself. It is this structuralresemblance that makes the skilled person expect some kind ofsynergistic action. Hence, at most the skilled person will regard theeffect of butylamine up to stearylamine linked to those specificantibiotic peptides only.

WO-A-00/74654 discloses an administration form containing an acid-labileactive compound in a matrix made of a mixture comprising triglycerideand solid paraffin. The mixture may contain further suitable excipients,such as polymers, sterols and basic compounds, among which stearylamine.Although WO-A-00/74654 suggests to combine the acid-labile activecompound with antibiotics, there is no disclosure of such anadministration form further containing antibiotics.

Similarly, U.S. Pat. No. 6,479,540 also teaches the use of stearylamineas a carrier. In the present case, the compositions containstocol-soluble therapeutics, including antibiotics, as the activeingredients. Stearylamine is one of the many candidates to form an ionpair with the active ingredient, in order to increase its tocolsolubility. No actual combination of an antibiotic and stearylamine isreported.

WO-A-00/30611 relates to compositions for treating protozoa, especiallycausative agents of malaria. It describes lipid vesicles which containstearylamine, surrounding penicillin or tetracycline. Although no recipeis given, it suggests the use of large amounts of stearylamine, to formlipid vesicles. No hint on antibiotic resistance is given.

WO-A-04/00360 addresses the problem of developing resistance toantibiotics in treating dermatoses, and teaches the use of topicaltherapy. Stearylamine and dodecylamine are among the many possible basiccompounds capable of producing a pH of 8.0 or greater in the topicalformulation, thus acting as a skin permeation enhancer. Relatively largeamounts are necessary for this purpose.

DE 10245506 describes formulations in which an active agent, such as anantibiotic, is embedded in a matrix of phospholipids andpalmitoyl-D-glucuronide. The formulations are administered parenterallyor by inhalation. The examples show that the amount of phospholipidslargely exceeds that of the active ingredient.

SUMMARY OF THE INVENTION

It is an objective of the present invention to provide antimicrobialcompositions. It is further an objective to provide antimicrobialcompositions that are active towards bacteria that are resistant to oneor more antibiotics, in particular it is an objective to provideantimicrobial compositions that are active towards bacteria that areresistant to one or more antibiotics comprising the one or moreantibiotics to which the bacteria are resistant. It is further anobjective to provide antimicrobial compositions that have an enhancedactivity of one or more antibiotics towards bacteria as compared to theantibiotic(s) alone.

It was found that certain long-chain alkylamines in combination with anantibiotic were capable of rendering bacteria susceptible to theantibiotic, whereas the antibiotic without the certain long-chainalkylamine was much less or not at all active against the bacteria.

Furthermore, it was found that long-chain alkylamines already promoteantibiotic activity at concentrations significantly lower than thosereported in the prior art.

Thus the invention concerns antimicrobial compositions comprising along-chain amine of formula I or a physiologically acceptable saltthereof

whereinR₁ represents a linear or branched alkyl group comprising at least 7atoms in a straight chain, said alkyl group may comprise double ortriple bonds and may contain one or more substitutions, one or morecycloalkyl and/or aryl rings and may comprise one or more O, N and/or Satoms, and one of R₂ and R₃ is as is defined for R₁ and may be the sameas R₁, or R₂ and R₃ are different from R₁, and R₂ and R₃ may be the sameor different and represent a hydrogen or a lower alkyl group, said loweralkyl group may comprise double or triple bonds and may contain one ormore substitutions or cycloalkyl and/or aryl rings and may comprise oneor more O, N and/or S atoms, and the compositions further contain atleast one antibiotic.

DETAILED DESCRIPTION OF THE INVENTION

The present compositions comprise an antibiotic and a compound offormula I as defined above, wherein said compound of formula I acts as asensitizer, meaning that it renders microorganisms, in particularbacteria, susceptible to the action of the antibiotic or that it renderssaid microorganisms susceptible to the action of the antibiotic at alower concentration or dosage of the antibiotic. In particular it isfound that bacteria that are resistant to certain types of antibiotics,or at least not affected by antibiotics at acceptable dosages, noweffectively could be stopped proliferating or in fact killed atacceptable concentrations of the antibiotic when this antibiotic isadministered in the presence of a sensitizer according to formula I.

Although it is immediately clear from the remainder of the text and theterm “sensitizer”, it is emphasized that the compound according toformula I is an active ingredient in the composition. Hence, it iscompletely different from those situations where it serves as a carriermaterial, generally defined not to interact with other components of thecomposition. However, in the present case, the compound according toformula I is precisely included to enhance the activity of theantibiotic. This is reflected by the preferred (relative) amounts of thesensitizer.

R₁ in formula I represents a linear or branched alkyl group comprisingat least 7 atoms in a straight chain. In this context alkyl group meansthat it is a group containing carbon atoms. For instance, for thelipoamines butylamine, pentylamine and hexylamine as disclosed in JP57155954 no effect was found in our studies. It is understood that ifnot specified otherwise, C, O, N and S atoms in the alkyl group furthercomprise hydrogen atoms to properly satisfy the valency of therespective atom. At least 7 atoms in a straight chain is with respect tothe longest group of atoms, not including hydrogen, directly connectedto one another by covalent bonds starting from the nitrogen (N) informula I. Such a group of at least 7 atoms in a straight chain thus canbe part of cyclic elements, including aromatic rings, in R₁, or in otherwords, one or more (saturated and/or unsaturated) rings can form (partof) the alkyl group comprising at least 7 atoms in a straight chain. Thealkyl group may comprise one or more O, N and/or S atoms, which meansthat the chain of carbon atoms may be interrupted by one or more O, Nand/or S atoms. It is preferred the alkyl group comprises an ω-CH₃group, or in other words starting from the nitrogen (N) in formula I thelongest alkyl group preferably ends with a CH₃.

The R₁ alkyl group, in particular the at least 7 atoms containingstraight chain, may comprise one or more double bonds or one or moretriple bonds or combinations of double and triple bonds and/orcycloalkyl and/or aryl rings. In one embodiment the at least 7 atomscontaining straight chain comprises one double bond.

Also the at least 7 atoms containing straight chain may be interruptedby one or more O, N and/or S atoms, yielding e.g. alkoxyalkyl,alkylthioalkyl, hydroxyalkyl, thioalkyl, aminoalkyl moieties and thelike.

Also the at least 7 atoms containing straight chain may be substituted,for example by one or more halogen atoms and/or groups via one or moreO, N and/or S atoms, e.g. hydroxyl, amine and/or thiol groups or O-, N-and/or S-lower (carboxy)alkyl or doubly bound O, N(—H or -alkyl) and/orS. Lower alkyl preferably means a group comprising 1-6 carbon atoms.

In one embodiment the at least 7 atoms containing straight chain alkylgroup only contains carbon atoms. In one embodiment the straight chainalkyl group contains 7-30 carbon atoms, preferably 10-24 carbon atoms,more preferably 12-22 carbon atoms, most preferably at least 13 carbonatoms. In one embodiment the R₁ alkyl group only contains carbon atoms.

In one embodiment only one relatively large alkyl group, i.e. an atleast 7 atoms containing straight chain alkyl group, is present in thesensitizer of formula I. In one embodiment R₂ and R₃ are different fromR₁. It is thus preferred that R₂ and R₃ are selected from the groupconsisting of hydrogen and lower alkyl, wherein lower alkyl preferablyis a group containing 1-6 carbon atoms.

The lower alkyl group may comprise one or more double bonds or one ormore triple bonds or combinations of double and triple bonds and/orcycloalkyl and/or aryl rings.

Also the lower alkyl group may be interrupted by one or more or end withO, N and/or S atoms, e.g. alkoxyalkyl, alkylthioalkyl, hydroxyalkyl,thioalkyl, aminoalkyl and the like.

Also the lower alkyl group may be substituted, for example by one ormore halogen atoms and/or groups via one or more O, N and/or S atoms,e.g. hydroxyl, amine and/or thiol groups or O-, N- and/or S-lower(carboxy)alkyl or doubly bound O, N(—H or -alkyl) and/or S. In oneembodiment lower alkyl group means C1-C6 alkyl.

In one embodiment R₂ and R₃ are different and at least one of R₂ and R₃is hydrogen. In another embodiment R₂ and R₃ are the same and preferablyare selected from the group consisting of C1-C4 alkyl, preferablymethyl, ethyl, propyl, isopropyl and butyl. In another embodiment R₂ andR₃ are the same and both are hydrogen.

In one embodiment the sensitizer of formula I is present in the form ofa salt, in particular an acid addition salt, e.g. its HCl, HBr, HF,H₃PO₄, H₂SO₄, citric acid, acetic acid, trifluoroacetic acid, lacticacid, isethionic acid, methanesulfonic acid or ethylenediaminetetraacetic acid addition salt.

In the present compositions, preferably the sensitizer of formula I ispresent in an amount that is sufficient to enhance the effectiveness ofthe antibiotic.

By an effectiveness of an antibiotic is understood that the addition ofthe antibiotic to a culture medium inhibits growth of the inoculum suchthat the number of colony forming units (CFU) with the antibiotic isless than 30%, such as 20%, 15%, 10%, or 5% of the CFU without additionof the antibiotic. Preferably the addition of the antibiotic kills theinoculum such that the CFU is less than 70%, such as 60%, 50%, 40%, 30%,20%, 10%, 5%, 2%, 1%, 0.1%, or 0.01% of the inoculum.

By an enhanced effect of an antibiotic is understood that the minimuminhibitory concentration (MIC) of the antibiotic without the sensitizerof formula I according to the present invention is decreased by at least2-fold by the addition of said sensitizer. Preferably, the decrease isat least 4-fold, such as 8-fold, 10-fold or even more such as 20-fold,50-fold or even 100-fold.

A person skilled in the art is able, based on routine experimentation,to determine what a suitable concentration of the sensitizer is, takinginto account the particular sensitizer, the particular antibiotic andthe extent to which enhanced effectiveness of the antibiotic isattained.

As reported above, it is part of the invention that the amounts ofsensitizer need not be high to enhance the effectiveness of theantibiotic(s). It is preferred that the molar ratio of sensitizer(s) offormula I to antibiotic(s) is attractively lower than 5:1, morepreferably lower than 2:1, most preferably lower than 1.5:1,particularly at most 1:1. More preferably, the molar ratio is at least1×10⁻⁵:1, more preferably at least 5×10⁻⁵:1, most preferably at least1×10⁻⁴:1.

Thus also the present compositions preferably comprise anantimicrobially, or antibiotically, effective amount of an antibiotic.From the above it follows that a skilled person is able, based onroutine experimentation, to determine what a suitable concentration ofthe antibiotic is, taking into account the particular sensitizer, theparticular antibiotic and the extent to which enhanced effectiveness ofthe antibiotic is attained.

In one embodiment the present composition comprises an antibiotic thatis effective against Gram-negative bacteria. Gram-positive andGram-negative bacteria are differentiated by the Gram stain. AGram-positive species retains the primary stain (crystal violet) whentreated with a decolourising agent (alcohol or acetone) whereas aGram-negative bacterium loses the primary stain. The staining differencereflects the structural differences in the cell walls of Gram-negativeand Gram-positive bacteria. The Gram-positive cell wall consists of arelatively thick peptidoglycan layer and teichoic acids whereas theGram-negative cell wall consists of a relatively thin peptidoglycanlayer, and an outer membrane consisting of a lipid bilayer containingphospholipids, lipopolysaccharide, lipoproteins and proteins.

In yet another embodiment the present composition comprises anantibiotic that is effective against Gram-positive bacteria.

In one embodiment the present composition comprises an antibiotic whichis selected from the group consisting of β-lactams, (e.g. ampicillin,ceftazidime, meropenem), quinolones (e.g. norfloxacin, ciprofloxacin),glycopeptides (e.g. vancomycin), macrolides (e.g. erythromycin),oxazolidinones (e.g. linezolid), peptide antibiotics (e.g. magainin II),lipopeptides (e.g. polymyxins, bacitracin), nitroimidazoles (e.g.metronidazole), ansamycins (e.g. rifampin), azoles (e.g. fluconazole),D-cycloserine, lincosamides (e.g. clindamycin), mupirocin,streptogramins (e.g. dalfopristin, quinupristin), fosfomycin,aminoglycosides (e.g. gentamicin), sulfonamides (e.g. sulfomethoxazole),trimethoprim, tetracyclines (e.g. tigilcycline), novobiocin,chloramphenicol, monobactams and synthetic derivatives of theseantibiotics.

More in particular, a (combination of) suitable antibiotic(s) to be usedin combination with the sensitizer according to formula I is selectedfrom the group consisting of glycopeptides (preferably vancomycin orteicoplanin), β-lactams, preferably penicillins, such as amdinocillin,ampicillin, amoxicillin, azlocillin, bacampicillin, benzathinepenicillin G, carbenicillin, cloxacillin, cyclacillin, dicloxacillin,methicillin, mezlocillin, nafcillin, oxacillin, penicillin G, penicillinV, piperacillin, and ticarcillin; cephalosporins, such as the firstgeneration drugs cefadroxil, cefazolin, cephalexin, cephalothin,cephapirin, and cephradine, the second generation drugs cefaclor,cefamandole, cefonicid, ceforanide, cefoxitin, and cefuroxime, or thethird generation cephalosprins cefoperazone, cefotaxime, cefotetan,ceftazidime, ceftizoxime, ceftriaxone, and moxalactam; carbapenems suchas imipenem; or monobactams such as aztreonam; further tetracyclinessuch as demeclocycline, tigilcycline, doxycycline, methacycline,minocycline, and oxytetracycline; aminoglycosides such as amikacin,gentamicin, kanamycin, neomycin, netilmicin, paromomycin, spectinomycin,streptomycin, and tobramycin; polymyxins such as colistin,colistimathate, and polymyxin B, and erythromycins and lincomycins andalso sulfonamides such as sulfacytine, sulfadiazine, sulfisoxazole,sulfamethoxazole, sulfamethizole, and sulfapyridine; trimethoprim,quinolones, novobiocin, pyrimethamine, and rifampin are also expected tohave enhanced activity in the presence of the sensitizer of formula I ina composition according to the present invention.

It is stressed that the sensitizer of the invention is not selected forits structural similarities with the antibiotic. It is found that thecompound according to formula I also enhances the antimicrobial effectof the aforementioned antibiotics other than peptide antibiotics andlipopeptides, although having little in common structurally.

The invention also concerns a composition of a sensitizer of formula Ias defined above and an antibiotic together with a pharmaceuticalacceptable carrier. Such a pharmaceutical composition may be in solid,semi-solid, liquid etc. form, which are for internal or externalapplication such as a tablet, capsule, liquor, vapour, ointment, paste,spray etc. Formulation into a suitable form is well known to a personskilled in the art, see e.g., “Remington's Pharmaceutical Sciences” and“Encyclopedia of Pharmaceutical Technology”.

For optimal interaction with the antibiotic, it is preferred that thesensitizer of formula I is not contained in a protective or embeddinglayer. The sensitizer of formula I and the antibiotic are preferablypresent in the same matrix.

Particularly preferred embodiments of the present invention are thosewherein the sensitizer of formula I is selected from the groupconsisting of 3-lauryloxypropylamine, laurylamine, myristylamine,palmitylamine, stearylamine, eicosamine, oleylamine, linoleylamine,linolenylamine, sphingosine. In another preferred embodiments thecompositions according to the present invention comprise the HCl salt of3-lauryloxypropylamine, laurylamine, myristylamine, palmitylamine,stearylamine, eicosamine, oleylamine, linoleylamine, linolenylamine,sphingosine, dehydroabietylamine, or the citrate salt of tamoxifen. Moreparticularly, the present invention comprises 3-lauryloxypropylamine,myristylamine, palmitylamine, eicosamine, oleylamine, linoleylamine,linolenylamine, sphingosine, dehydroabietylamine, tamoxifen, or the HClsalt thereof, or the citrate salt of tamoxifen.

In general the present compositions can be used to eradicate Gramnegative and/or Gram positive bacteria from places where they are notdesired. Thus the present invention also concerns the use of the presentantimicrobial compositions for cleaning or sterilising of objects andareas. Preferably for this purpose the sensitizers of formula I asdefined above and (an) antibiotic(s) and optionally further cleaningand/or sterilising agents are combined with a suitable carrier ordiluent, for example such as water and/or (an) alcohol.

Animal feed formulations are not a preferred embodiment of theinvention. It is more preferred that the composition of the invention isa pharmaceutical composition.

It is not part of the invention to provide novel administration formsfor acid-labile active compounds. The composition of the invention ispreferably free from such acid-labile active compounds, such asacid-labile proton pump inhibitors (H+/K+ ATPase inhibitors), inparticular substituted pyridine-2-yl-methylsulfinyl-1H-benzimidazoles orprazoles. Alternatively or additionally, it is preferred not to usesolid paraffin, such as paraffinum solidum (paraffin wax) or ozoceritein the composition.

The present invention also concerns the use of sensitizers of formula Ias defined above and (an) antibiotic(s) for the preparation of amedicament for the treatment of a bacterial infection, preferably inhuman beings, particularly to enhance the antimicrobial activity of theantibiotic(s). Alternatively, the present invention also provides amethod for treating bacterial infections in a patient in need thereof,preferably a human being, the method comprising administering thecomposition of the invention to the patient.

In one embodiment the medicament is used for the treatment of infectionsby bacteria that are resistant or multi-resistant to certain specificantibiotics. In one embodiment the medicament is used for the treatmentof infection by Gram-negative bacteria. In one embodiment the medicamentis used for the treatment of infections by Escherichia spp, inparticular E. coli, Haemophilus spp, in particular H. influenzae,Pseudomonas spp, in particular P. aeruginosa, Klebsiella, in particularK. pneumoniae , Enterobacter spp., Helicobacter spp, in particularHelicobacter pylori, Shigella spp, Salmonella spp, Yersinia spp,Campylobacter spp, Neisseria spp, Bordetella spp, Aeromonas spp,Burkholderia spp, Serratia spp, Vibrio spp, Proteus mirabilis, andAcinetobacter spp, in particular A. baumannii.

In one embodiment the medicament is used for the treatment of infectionby Gram-positive bacteria. In one embodiment the medicament is used forthe treatment of infections by Staphylococcus spp, in particularmethicillin-resistant Staphylococcus aureus, Streptococcus spp,Enterococcus spp, Listeria spp, Staphylococcus spp, Streptococcus spp,Clostridium spp, Bacillus spp, Enterococcus spp, Corynebacterium spp,Legionella spp, Mycobacterium spp,

Additionally, the invention also pertains to a kit of parts comprising:

-   -   a) at least one sensitizer represented by formula I; and    -   b) at least one antibiotic,        intended for the treatment of a bacterial infection,        particularly to enhance the antimicrobial effect of the        antibiotic.

The sensitizer and the antibiotic may comprise one or more additionalfeatures as defined above. The kit of parts is intended for sequentialor simultaneous administration, wherein the administration routes forthe sensitizer and the antibiotic may be the same or different. Therein,it is preferred to adapt the amount of sensitizer administered toenhance the effectiveness of the antibiotic. Means for achieving thisare described above.

EXAMPLES Experimental Procedure for Assaying Sensitizers of Formula I

Solution

An 8 mM stock solution of long-chain alkylamine of formula I in ethanolwas prepared for serial 2-fold dilution with ethanol. One equivalent ofhydrochloric acid (from 1 M aqueous stock) was added to the firstsolution before dilution, in case the amine was not in the HCl-form.Tamoxifen citrate was dissolved in ethanol to provide the first 8 mMstock solution.

Bacteria

The concentration of an overnight culture (16 h, 37° C.) of Pseudomonasaeruginosa PA01 in 100% LB was determined by comparison with acalibration curve and diluted to 1×10⁵ CFU/mL with 100% LB. Likewise,cultures of the following clinical isolates (obtained at the LeidenUniversity Medical Center) were prepared: multidrug-resistantAcinetobacter baumannii LUH5771, extended β-lactamase (ESBL) producingKlebsiella pneumoniae LUH5344 and Pseudomonae aeruginosa PA7243, PA7247,PA7249, PA7252, PA7253 and PA 24-7-3 (ceftazidime-resistant).

Test

Using 96-well plates, concentrations of sensitizer (1 μL of everyethanolic solution) were added to the wells containing 20 μL P.aeruginosa (final concentration 1×10⁴ CFU/mL, OD₅₅₀=0.1) and ampicillinor linezolid (in cases of PA 24-7-3 and ESBL K. pneumoniae LUH5344,ceftazidime was used). For 1×10⁴ CFU/mL A. baumannii LUH5771, the effectof sensitizers on growth was investigated in presence of gentamicin. Thevolume in the wells was adjusted to 200 μL with 20% LB. As controls, 20%LB, bacteria+20% LB, bacteria+20% LB+antibiotic were included. In somecases the medium was changed to 100% LB (vide infra) After addition ofsensitizer at t=0, the 96-well plate was covered (not airtight) andincubated at 37° C. while shaking for 20 h in a BioTek plate reader;OD₅₅₀ was determined at least every 10 min for PA01 studies or onceafter 20 h for clinical isolates. A MIC value for a specific compoundwas determined as the lowest concentration at which, after 20 hincubation, the OD₅₅₀ value was comparable to that of the blank used.

Results

TABLE 1 Effect of sensitzers on the growth of P. aeruginosa PA01 in 20%LB in presence of ampicillin or linezolid. Compounds Effect ampicillinMIC >200 μg/mL linezolid MIC >200 ug/mL oleylamine MIC at ≧20 μM 50μg/mL ampicillin + oleylamine MIC at ≧0.31 μM oleylamine 200 μg/mLampicillin + oleylamine MIC at ≧0.08 μM oleylamine 100 ug/mL linezolid +oleylamine MIC at ≧5 μM oleylamine stearylamine MIC at ≧20 μM 200 μg/mLampicillin + stearylamine MIC at ≧0.62 μM stearylamine 100 ug/mLlinezolid + stearylamine MIC at ≧10 μM stearylamine dehydroabietylamineMIC at ≧20 μM 200 μg/mL ampicillin + MIC at ≧10 μM dehydroabietylaminedehydroabietylamine tamoxifen MIC at ≧20 μM 200 μg/mL ampicillin +tamoxifen MIC at ≧2.5 μM tamoxifen

Representative examples of the sensitizers of formula I are oleylamineand stearylamine. As is shown in Table 1, these compounds render P.aeruginosa PA01 vulnerable to ampicillin. PA01 was killed by 200 μg/mLampicillin in the presence of 0.62 μM stearylamine or 0.08 μMoleylamine. The growth of PA01 was also remarkably inhibited bylinezolid in presence of sensitizer; linezolid is an antibiotic that isindicated only for treatment of Gram-positive species. PA01 was not onlyinhibited by ampicillin in combination with fatty amines, but also bycombining ampicillin with the tricyclic dehydroabietylamine oranti-cancer agent tamoxifen.

Similarly, the effects of oleylamine as sensitizer were determinedagainst clinical isolates of P. aeruginosa (Table 2). These isolates canbe eradicated by the combination of ampicillin+oleylamine while beingunaffected to high concentrations of both.

TABLE 2 Effect of oleylamine on the growth of clinical P. aeruginosaisolates in presence of ampicillin. Growth inhibiting (MIC) MICcompositions of ampicillin at Isolate ampicillin oleylamineconcentrations oleylamine PA7243 >200 μg/mL MIC at >40 μM 200 μg/mL at≧5 μM  50 μg/mL at ≧7.5 μM PA7247 >200 μg/mL MIC at ≧40 μM 200 μg/mL at≧7.5 μM  50 μg/mL at ≧10 μM PA7249 >200 μg/mL MIC at >40 μM 200 μg/mL at≧2.5 μM  50 μg/mL at ≧7.5 μM PA7252 >200 μg/mL MIC at ≧40 μM 200 μg/mLat ≧1.25 μM  50 μg/mL at ≧5 μM PA7253 >200 μg/mL MIC at >40 μM 200 μg/mLat ≧5 μM  50 μg/mL at ≧10 μM Inoculum 10⁴ bacteria in 20% LB, 20 hincubation.

Table 3 displays the effects of low concentrations of the sensitizersoleylamine and stearylamine on the MIC values of ampicillin to whichPA01 normally is resistant; furthermore, Table 3 shows that the MICvalue of the quinolone antibiotic nalidixic acid against PA01 is reducedupon addition of low concentrations of sensitizer. It should be notedthat the MIC value of 25 μg/mL of an antibiotic is classified asclinically resistant.

TABLE 3 Effect of sensitzers on the MIC value of antibiotics against P.aeruginosa PA01 (after 20 h). MIC (μg/mL) ampicillin >200 ampicillin +oleylamine 5 μM 3.125 ampicillin + stearylamine 3 μM 6.25 nalidixic acid25 nalidixic acid + oleylamine 5 μM 12.5 nalidixic acid + oleylamine 20μM 6.3 Inoculum 10⁴ bacteria in 20% LB.

Furthermore, a multi-resistant Acinetobacter baumannii clinical isolatewas found to be vulnerable to the action of gentamicin in the presenceof oleylamine, whereas the species itself was resistant to treatmentwith gentamicin, ampicillin, the frequently used combination ofgentamicin/ampicillin, or oleylamine alone (see Table 4).

TABLE 4 Effect of sensitizers on susceptibility of a multi-drugresistant A. baumannii towards gentamicin. MIC MIC of gentamicin atgentamicin oleylamine concentration oleylamine AC >200 μg · mLbactericidal 100 μg/mL at ≧0.62 μM LUH5771 at ≧5 μM

Finally, use of oleylamine as sensitizer could lower the MIC value ofceftazidime significantly in cases of an extended β-lactamase producingK. pneumoniae and P. aeruginosa clinically isolated strains.

TABLE 5 Effect of compositions on extended-β-lactamase producing K.pneumoniae clinical isolate LUH5344 and ceftazidime-resistant P.aeruginosa clinical isolate PA 24-7-3. MIC MIC ceftazidime + ceftazidimeoleylamine oleylamine ESBL 6.25 μg/mL MIC ≧40 μM MIC <0.098 μg/mLLUH5344 at 20 μM oleylamine PA 24-7-3 6.25 μg/mL MIC ≧20 μM MIC 1.56μg/mL at 10 μM oleylamine (100% LB (LUH5344) or 20% LB (PA 24-7-3))

Similar results as depicted in the Tables above could be obtained usingsensitizers with shorter R₁ groups (as depicted in formula I), such aslaurylamine.

In the same manner as described above also methicillin-resistantStaphylococcus aureus (MRSA) is tested and is found to be sensitivetowards methicillin when this is administered together with thelong-chain alkylamines described above such as oleylamine andstearylamine. Likewise, vancomycin-resistant Enterococcus faecalis (VRE)can be killed with vancomycin and other glycopeptides in presence ofsensitizers of formula I. Also, lower antibiotic dosages are found to benecessary for inhibiting growth of Staphylococcus spp, Streptococcusspp, Clostridium spp, Bacillus spp, Enterococcus spp, Corynebacteriumspp, Legionella spp, Mycobacterium spp, Listeria spp in presence ofsensitizers of formula I.

1. An antimicrobial composition comprising a) a long-chain alkylamine offormula I or a physiologically acceptable salt thereof

wherein R₁ represents a linear or branched alkyl group comprising atleast 7 atoms in a straight chain, said alkyl group may comprise doubleor triple bonds and may contain one or more substitutions, cycloalkyl oraryl rings, and may comprise one or more O, N and/or S atoms, and R₂ andR₃ may be the same or different and represent a hydrogen or a loweralkyl group comprising 1-6 carbon atoms, said lower alkyl group maycomprise double or triple bonds, cycloalkyl or aryl rings, and maycontain one or more substitutions and may comprise one or more O, Nand/or S atoms, and b) at least one antibiotic, wherein the molar ratioof a) to b) is lower than 5:1.
 2. The antimicrobial compositionaccording to claim 1, wherein said molar ratio of a) to b) is at most1:1.
 3. The antimicrobial composition according to any one of thepreceding claims, wherein a) and b) are present in the same matrix. 4.The antimicrobial composition according to any one of the precedingclaims, being a pharmaceutical composition.
 5. The antimicrobialcomposition according to any one of the preceding claims, wherein the atleast 7 atoms containing straight chain alkyl group only contains carbonatoms.
 6. The antimicrobial composition according to any one of thepreceding claims, wherein the straight chain alkyl group contains 12-22carbon atoms.
 7. The antimicrobial composition according to any one ofthe preceding claims, wherein the R₁ alkyl group only contains carbonatoms.
 8. The antimicrobial composition according to any one of thepreceding claims, wherein R₂ and R₃ represent hydrogen.
 9. Theantimicrobial composition according to any one of the preceding claims,wherein the long-chain alkylamine of formula I is in the form of its HClsalt.
 10. The antimicrobial composition according to any one of thepreceding claims, wherein the long chain amine is selected from thegroup consisting of laurylamine, myristylamine, palmitylamine,eicosamine, oleylamine, sphingosine, 3-lauryloxypropylamine,linoleylamine, linolenylamine, dehydroabietylamine, tamoxifen.
 11. Theantimicrobial composition according, to any one of the preceding claims,wherein the antibiotic is selected from β-lactams, quinolones,glycopeptides, macrolides, oxazolidinones, peptide antibiotics,lipopeptides, nitroimidazoles, ansamycins, azoles; D-cycloserine,-lincosamides, mupirocin, streptogramins, fosfomycin, aminoglycosides,sulfonamides, trimethoprim, tetracyclines, novobiocin, chloramphenicol,monobactams and synthetic derivatives thereof.
 12. Use of a compositionaccording to any one of the preceding claims for the preparation of amedicament for the treatment of a bacterial infection.
 13. Use accordingto claim 12, to enhance the antimicrobial activity of the antibiotic(s)14. Use according to claim 12 or 13 for the treatment of infection byGram-negative bacteria.
 15. Use according to claim 12-14 for thetreatment of infections by Escherichia spp, Haemophilus spp, Pseudomonasspp, Klebsiella spp, Enterobacter spp., Helicobacter spp, Shigella spp,Salmonella spp, Yersinia spp, Campylobacter spp, Neisseria spp,Bordetella spp, Aeromonas spp, Burkholderia spp, Serratia spp, Proteusspp, Vibrio spp and Acinetobacter spp.
 16. Use according to claim 12 forthe treatment of infection by Gran-positive bacteria.
 17. Use accordingto claim 12 or 16 for the treatment of infections by Staphylococcus spp.Streptococcus spp, Clostridium spp, Bacillus spp, Enterococcus spp,Corynebacterium spp, Legionella spp, Mycobacterium spp, and Listeriaspp.
 18. Use of the antimicrobial composition according to any one ofclaims 1-11, optionally comprising further cleaning and/or sterilisingagents and optionally comprising a suitable carrier or diluent, forcleaning or sterilising of objects and areas.
 19. Kit of partscomprising: a) a long-chain alkylamine of formula I or a physiologicallyacceptable salt thereof

wherein R₁ represents a linear or branched alkyl group comprising atleast 7 atoms in a straight chain, said alkyl group may comprise doubleor triple bonds and may contain one or more substitutions, cycloalkyl oraryl rings, and may comprise one or more O, N and/or S atoms, and R₂ andR₃ may be the same or different and represent a hydrogen or a loweralkyl group comprising 1-6 carbon atoms, said lower alkyl group maycomprise double or triple bonds, cycloalkyl or aryl rings, and maycontain one or more substitutions and may comprise one or more O, Nand/or S atoms, and b) at least one antibiotic, wherein the molar ratioof a) to b) is lower than 5:1.
 20. Use of the kit of parts according toclaim 19 for the preparation of a medicament for the treatment of abacterial infection.